Expression and Purification of Hybrid LL-37Tα1 Peptide in Pichia pastoris and Evaluation of Its Immunomodulatory and Anti-inflammatory Activities by LPS Neutralization

Abstract

This study pertains to the new approach for the development of hybrid peptide LL-37Tα1 and its biomedical applications. A linear cationic hybrid peptide, LL-37Tα1 was derived from two parental peptides (LL-37 and Tα1) recognized as potent anti-endotoxin without any hemolytic or cytotoxic activity. We successfully cloned the gene of hybrid peptide LL-37Tα1 in PpICZαA vector and expressed in the Pichia pastoris. The recombinant peptide was purified by Ni-affinity column and reverse-phase high performance liquid chromatography (RP-HPLC) with an estimated molecular mass of 3.9 kDa as determined by SDS-PAGE and mass spectrometry. We analyzed the LPS neutralization by limulus amebocyte lysate (LAL) activity and the results indicate that the hybrid peptide LL-37Tα1 directly binds endotoxin and significantly (p < 0.05) neutralizes the effect of LPS in a dose-dependent manner. Lactate dehydrogenase (LDH) assay revealed that LL-37Tα1 successfully reduces the LPS-induced cytotoxicity in mouse RAW264.7 macrophages. Moreover, it significantly (p < 0.05) decreased the levels of nitric oxide, proinflammatory cytokines including TNF-α, IL-6, IL-1β, and diminished the number of apoptotic cells in LPS-stimulated mouse RAW264.7 macrophages. Our results suggest that the P. pastoris expression system is cost-effective for commercial production of the immunomodulatory and anti-inflammatory hybrid peptide (IAHP) LL-37Tα1 and the peptide may serve as effective anti-endotoxin/anti-inflammatory agent with minimal cytotoxicity.

Keywords: LPS neutralization; apoptosis; hybrid peptides; immunomodulatory; yeast expression.

Figure 1

Construction of recombinant plasmid pPICZαA-LL-37Tα1.

Figure 2

Tricine-SDS-PAGE and analysis of recombinant peptide, (A) Tricine-SDS-PAGE of the cell culture media from P.pastoris expressing secreted LL-37Tα1. Lane M, molecular weight markers; Lane C, control (supernatant of X33/PpICZαA); Lane 1 and 2 (supernatant X33/PpICZαA-LL-37Tα1) peptide expression after methanol (144 h) induction and arrow in the lane indicated 3.9 kDa polypeptide. (B) Tricine-SDS-PAGE of recombinant purified LL-37Tα1. Lane M, molecular weight markers; Lane C, control (X33/PpICZαA); Lane 1, a sample of the purified recombinant peptide and arrow indicated LL-37Tα1 (3.9 kDa) expression. (C) The elution pattern of RP-HPLC C18 column of the purified recombinant LL-37Tα1 and the high peak indicates fraction that contains LL-37Tα1. (D) ESI-MS analysis of purified recombinant LL-37Tα1 peptide.

Figure 3

LPS neutralization, cytotoxicity, and hemolytic activity of parental and recombinant LL-37Tα1 peptide. (A) LPS neutralization by LL-37 and LL-37Tα1 determined using an endotoxin quantitation kit. Mean values presented; n = 3 ± SD (^#p < 0.05 and ^##p < 0.01 showed comparison of LL-37 vs. LL-37Tα1). (B) LL-37αT1 peptide decreased cytotoxicity in the cultured medium of LPS-infected mouse RAW264.7 macrophages. Data represented as mean ± standard deviation (SDs) of independent experiments. ^*p < 0.05,^**p < 0.01,^***p < 0.001, vs. LPS. Whereas, ^#p < 0.05 and ^##p < 0.01 indicates significant difference compared with parental LL-37 peptide. (C) Hemolytic activities of LL-37Tα1 against mouse RBCs. The data correspond to the mean values of three independent experiments and are expressed as a percentage of hemolysis ± standard deviation (^***p < 0.001 vs. Triton X-100). While, ^#p < 0.05 and ^##p < 0.01 indicates significant difference compared with parental LL-37 peptide.

Figure 4

Effect of LL-37 and recombinant LL-37Tα1 peptide on LPS-induced inflammatory response in mouse RAW264.7 macrophages. (A) Nitric oxide (NO) production, (B) level of TNF-α, (C) IL-6, and (D) IL-1β, Cell were infected by LPS to stimulate inflammation and treated with various concentration of LL-37 and LL-37Tα1. After 24 h incubation, culture media were collected and inflammatory cytokines level were determined. Values are means ± SD of three independent experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, vs. LPS. While, ^#p < 0.05 and ^##p < 0.01 indicates significant difference compared with parental LL-37 peptide.

Figure 5

Apoptosis of mouse RAW264.7 macrophages treated with LPS alone and co-infection with parental and our hybrid peptide. Mouse RAW264.7 macrophages were treated with LPS and without LL-37 and LL-37Tα1+LPS at 4, 12, and 24 h with FITC-conjugated annexin-V (Green) and PI (Red). (A) The stained mouse RAW264.7 macrophages were examined by flow cytometry, Control represents normal cells, the middle panel LPS representative mouse RAW 264.7 macrophages treated with LPS only, and right panel LL-37+LPS, LPS+LL-37Tα1 reveals mouse RAW64.7 macrophages treated with LPS and peptide. (B) Percentile value of apoptotic cells after treatment of LPS alone or with a hybrid peptide with mouse RAW64.7 macrophages. ^*p < 0.05 and ^**p < 0.01 vs. LPS indicate the significance and highly significance difference. ^#p < 0.05 and ^##p < 0.01 indicates significant difference in hybrid peptide compared with parental LL-37 peptide.

Figure 6

Schematic diagram of the potential mechanism by which LL-37Tα1 suppresses LPS-induced inflammatory responses in mouse RAW264.7 macrophages. Left, LPS alone; right LPS plus LL-37Tα1. The LL-37Tα1 binds with LPS and neutralizing it. (↑), activation; (⊣), no activation; (↑), upper regulate responses; (↓), lower regulate responses.